Research Article |
Corresponding author: Yu-Feng Hsu ( t43018@ntnu.edu.tw ) Academic editor: Thomas Simonsen
© 2018 Li-Wei Wu, Wen-Jie Lin, Yu-Feng Hsu.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Wu L-W, Lin W-J, Hsu Y-F (2018) A cryptic species, Dodona formosana, detected in the Dodona eugenes species complex: clarification of the taxonomic status of the Dodona butterfly in Taiwan. ZooKeys 736: 59-77. https://doi.org/10.3897/zookeys.736.22062
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The Tailed Punch, Dodona eugenes, is widely distributed in East Asia with seven subspecies currently recognized. However, two of them, namely ssp. formosana and ssp. esakii found in Taiwan, are hard to distinguish from each other due to ambiguous diagnostic characters. In this study, their taxonomic status is clarified by comparing genitalia characters and phylogenetic relationships based on mitochondrial sequences, COI and COII (total 2211 bps). Our results show that there is no reliable feature to separate these two subspecies. Surprisingly we found that Dodona in Taiwan is more closely related to the Orange Punch, D. egeon, than to other subspecies of D. eugenes. Therefore, the following nomenclatural changes are proposed: Dodona eugenes formosana is revised to specific status as Dodona formosana Matsumura, 1919, stat. rev, and ssp. esakii is sunk to a junior synonym of Dodona formosana syn. n.
endemism, male genitalia, Myrsinaceae , wing pattern
Dodona eugenes Bates 1868 is a medium-sized metalmark butterfly, distributed in East Asia from Muri (Pakistan), Nepal, north India, Bhutan, Indochina, western China, Hainan to Taiwan. Seven subspecies were recognized (
The distribution of the two putative Dodona subspecies in Taiwan is still difficult to document based on literature (
The Dodona eugenes species complex was proposed for a few closely related species, which share similar wing markings as D. eugenes (
To verify taxonomic status of the Dodona butterfly in Taiwan, a total of 92 riodinid individuals was sampled for morphological and molecular analyses (Suppl. material
Abbreviations for depositories:
SEHU Systematic Entomology Laboratory, Faculty of Agriculture, Hokkaido University, Japan
In total, 65 riodinid specimens were examined for male genitalia morphology, including 27 spp. formosana, eleven spp. esakii, ten D. maculosa, five D. eugenes eugenes, four D. egeon, three D. adonira, one D. eu. venox, one D. ouida, one Takashia nana, and one Polycaena chauchawensis (Suppl. material
DNA was extracted from two legs or thorax muscle using the Puregene DNA Isolation kit (Gentra Systems, Minnesota, USA). Mitochondrial cytochrome c oxidase 1 (COI) and cytochrome c oxidase 2 (COII) genes were amplified using the primers listed in Supplementary file 2. Polymerase Chain Reaction (PCR) was performed in a 30 μL volume, containing 23.5 μL of sterile ddH2O, 1 μL of extracted DNA, 1 μL of 10 μM dNTP, 3 μL of 10X PCR reaction buffer, 0.6 μL of each 10 μM primer, and 0.3 μL of Power Taq (Genomics Biosci & Tech, Taiwan). PCR was carried out using two settings as follows: (1) Standard: initial denaturation of 5 mins at 95 °C, followed by 40 cycles consisting of denaturation of 30 s at 95 °C, annealing of 30 s at 57–47 °C, and extension of 30–60s at 72 °C, and final extension of 7 mins at 72 °C; (2) Touchdown: initial denaturation of 5 mins at 95 °C, followed by 20 cycles consisting of three steps of 30 s at 95 °C, 30 s at 65–55 °C (-0.5 °C per cycle), and 30 s at 72 °C, and then additional 20 cycles consisting of 30 s at 95 °C, 30 s at 55–45 °C, and 30 s at 72 °C, and final extension of 7 mins at 72 °C. The quality of PCR products were visually checked on 1–2% agarose gels. If DNA fragments were correctly amplified, the PCR products were sequenced using an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA).
DNA sequences were checked and corrected by eye using Sequencher 4.10 (Gene Codes, Ann Arbor, USA). Sequence matrices were aligned using MUSCLE (
Molecular phylogenies were reconstructed under the Bayesian inference (BI) and Maximum Likelihood (ML) criteria. The BI analysis was performed in MrBayes v. 3.2.6 (
Partitions and substitution models used in this study. Substitution models for ML method were reduced to GTR+G.
Method | Partition nos. | Partitions and substitution models |
Bayesian inference | 3 | COI position 1 + COII position 1 + COII position 2 (HKY+I), COI position 2 (F81), COI position 3 + COII position 3 (GTR+I) |
Maximum Likelihood | 3 | COI position 1 + COII position 1 (GTR+I+G), COI position 2 + COII position 2 (GTR+G), COI position 3 + COII position 3 (GTR+I+G) |
To examine the genetic structure of Dodona in Taiwan, a haplotype network was constructed using the TCS 1.21 software (
Comparing male genitalia of ssp. formosana (n = 27) and ssp. esakii (n = 11), no clear difference was recognized (Fig.
Morphological comparison of genitalia between ssp. formosana and ssp. esakii. A Shape of genitalia (t-test, t = -0.9868, d.f. = 18, p = 0.3368) B Valva (t-test, t = 2.9904, d.f. = 18, p < 0.05) C Uncus (t-test, t = 4.6152, d.f. = 18, p<0.001) D Phallus (t-test, t = 0.6356, d.f. = 18, p = 0.5331).
A total of 43 specimens was sequenced for COI and COII (Suppl. material
Haplotype network was inferred based on an aligned matrix (2211 bps) comprised by 19 Dodona individuals from Taiwan. There are 30 variable sites, with 15 haplotypes found in 14 localities. Haplotype A was found in four different localities, including sites in both northern and central Taiwan (Fig.
The Kimura-2-Parameter pairwise distance (K2P-distance) between species were calculated and shown on Table
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
---|---|---|---|---|---|---|---|---|---|
1. Dodona adonira | |||||||||
2. Dodona egeon | 0.073 | ||||||||
3. Dodona elvira | 0.093 | 0.079 | |||||||
4. Dodona eugenes | 0.079 | 0.052 | 0.087 | ||||||
5. Dodona formosana | 0.073 | 0.034 | 0.085 | 0.056 | |||||
6. Dodona maculosa | 0.080 | 0.043 | 0.089 | 0.057 | 0.045 | ||||
7. Dodona ouida | 0.082 | 0.081 | 0.095 | 0.082 | 0.080 | 0.088 | |||
8. Polycaena chauchawensis | 0.093 | 0.085 | 0.092 | 0.088 | 0.087 | 0.094 | 0.086 | ||
9. Polycaena princeps | 0.095 | 0.089 | 0.092 | 0.097 | 0.092 | 0.098 | 0.098 | 0.053 | |
10. Takashia nana | 0.089 | 0.084 | 0.087 | 0.082 | 0.084 | 0.092 | 0.083 | 0.061 | 0.071 |
Based on the evidence from genitalia morphology and phylogenetic relationships, Dodona from Taiwan should be separated from D. eugenes and regarded as a distinct species. We therefore raised the Dodona butterfly in Taiwan to full species status, Dodona formosana
Dodona eugenes var. formosana Matsumura, 1919: Thous. Ins. Jap. Vol. 3: 591, pl. 46, f. 4, 5 (Type locality: Shito and Tochosi, Taihoku Pref., North Formosa; Holotype in SEHU).
Dodona eugenes matsumurana Nomura, 1930: Zephrus 2(2): 157–159, pl. 8, f. 1 (Type locality: Oowaki, Chikuto, Shinchiku Pref.; Holotype in SEHU).
Balonca formosana Hirayama, 1939: Genshoku Chrôui Zufu: pl. 22, f. 1, 2 (Type locality: Urai, Taihoku pref., North Formosa).
Dodona eugenes esakii Shirôzu, 1952: Sieboldia 1(1): 23-24, pl. 8, f. 44, 45, 48, 49 (Type locality: Taikokei valley and Heiganzan, Taichû Pref.), syn. n.
Holotype (Fig.
Additional material. 1 ♂ labelled ‘2013. IX.24 (Collect.)’, ‘Mt. Erge Shiding Xinbei’, ‘leg. W. J. Lin, C. W. Huang, C. J. Peng, Y. T. Chen’; 1 ♂ labelled ‘2014. VII.03 (Collect.)’, ‘2014.IX.20 (Eclosion)’, ‘Mt. Erge Shiding Xinbei’, ‘leg. W. J. Lin’, ‘genitalia Dn066’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘2014.VII.09 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’, ‘genitalia Dn034’, ‘14E37’; 1 ♂ labelled ‘2014.VII.07 (Eclosion)’, ‘2014. V.17 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’, ‘14E37’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘2014.VII.09 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’, ‘genitalia lot. Dn038’, ‘14E37’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘2014.VII.09 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’, ‘genitalia lot. Dn039’, ‘14E37’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’; 1 ♂ labelled ‘2014. V.17 (Collect.)’, ‘2014.VII.09 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. K. W. Hsiao, Y. M. Hsu’, ‘genitalia lot. Dn033’, ‘14E37’; 1 ♂ labelled ‘2014. V.24 (Collect.)’, ‘2014.VII.15 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’, ‘14E50’; 1 ♂ labelled ‘2014. V.24 (Collect.)’, ‘2014.VII.15 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’, ‘14E50’; 1 ♀ labelled ‘2014.VII.09 (Eclosion)’, ‘2014. V.24 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’, ‘14E50’; 1 ♀ labelled ‘2014. V.24 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’, ‘14E48’; 1 ♀ labelled ‘2014. V.24 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’, ‘14E48’; 1 ♂ labelled ‘2014. V.24 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin, C. P. Hseuh’; 1 ♂ labelled ‘2014. VIII.03 (Collect.)’, ‘Mt. Erge Shiding Xinbei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn023’; 1 ♂ labelled ‘2014. VIII.03 (Collect.)’, ‘2014.IX.25 (Eclosion)’, ‘Mt. Erge Shiding Xinbei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn024’; 1 ♂ labelled ‘2014. VIII.03 (Collect.)’, ‘2014.IX.21 (Eclosion)’, ‘Mt. Erge Shiding Xinbei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn025’; 1 ♂ labelled ‘2015. VI.27 (Collect.)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn067’; 1 ♂ labelled ‘2015. VI.27 (Collect.)’, ‘2015.VIII.17 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn064’; 1 ♂ labelled ‘2015. VIII.04 (Collect.)’, ‘2015.IX.20 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn065’; 1 ♂ labelled ‘2015. VIII.04 (Collect.)’, ‘2015.IX.24 (Eclosion)’, ‘Daluntou Neihu Taipei’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn082’; 1 ♂ labelled ‘2014.VIII.30 (Collect.)’, ‘Mt. Neinaotsui Jianshi Xinchu’, ‘leg. L. H. Wang, J. Y. Liang’, ‘genitalia lot. Dn032’, ‘14H41-1-MA03’; 1 ♂ labelled ‘2014.VIII.30 (Collect.)’, ‘Mt. Neinaotsui Jianshi Xinchu’, ‘leg. L. H. Wang, J. Y. Liang’, ‘genitalia lot. Dn070’, ‘14H41-2-MS01’; 1 ♂ labelled ‘2013.VII.27 (Eclosion)’, ‘2013.VI.20 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’; 1 ♀ labelled ‘2013.VIII.02 (Eclosion)’, ‘2013.VI.22 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’; 1 ♂ labelled ‘2013.VIII.10 (Eclosion)’, ‘2013.VI.25 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn027’; 1 ♂ labelled ‘2013.IX.29 (Eclosion)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn010’; 1 ♂ labelled ‘2013. XI.08 (Eclosion)’, ‘2013.XI.29 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn026’; 1 ♂ labelled ‘2014.III.16 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn028’; 1 ♂ labelled ‘2014. IV.01 (Eclosion)’, ‘2014.III.16 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin’, ‘genitalia lot. Dn028’; 1 ♂ labelled ‘2014. X.07 (Eclosion)’, ‘2014.XI.20 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin, C. C. Lin’, ‘genitalia lot. Dn083’; 1 ♂ labelled ‘2014. IV.02 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin, C. J. Chang, Y. H. Lin, M. F. Chou’, ‘genitalia lot. Dn059’; 1 ♂ labelled ‘2014. IV.02 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin, C. J. Chang, Y. H. Lin, M. F. Chou’, ‘genitalia lot. Dn084’; 1 ♂ labelled ‘2014.IX.28 (Collect.)’, ‘2014. VIII.25 (Eclosion)’, ‘Songmao Forest Road Hoping’, ‘leg. W. J. Lin, C. W. Huang, Y. M. Hsu, Y. H. Lin’, ‘genitalia lot. Dn085’; 1 ♂ labelled ‘2014. XI.11 (Eclosion)’, ‘2014.X.07 (Collect.)’, ‘Mt. Malun Hoping Taichung’, ‘leg. W. J. Lin, C. C. Lin’, ‘genitalia lot. Dn068’; 1 ♂ labelled ‘2015.VII.05 (Collect.)’, ‘Taiwan No. 8 Highway 119.5K Xiulin Hualien’, ‘leg. L. Huang’, ‘genitalia lot. Dn062’, ‘DNA lot. Rd030’.
Pinned specimens of Dodona formosana and their relatives. A D. formosana (spp. formosana) ♂ B D. formosana (spp. formosana) ♀ C D. formosana (spp. esakii) ♂ D D. formosana (spp. esakii) ♀ E D. eu. eugenes ♂ F D. eu. venox ♂ G D. egeon ♂ H D. egeon ♀ I D. maculosa ♂ J D. maculosa ♀ K type of D. formosana (SEHU).
Male (Fig.
Female (Fig.
Wing pattern of Dodona formosana is similar to D. eugenes and D. maculosa Leech 1890, in sharing small markings and narrow stripes (Fig.
Male genitalia of D. formosana is quite different from that of D. eugenes and D. maculosa, especially in the following characters (Fig.
Eggs are laid singly, or a few in a small cluster on the leaf or branch, also in debris near hostplant. Larval hostplants are M. seguinii and M. africana. The 1st and 2nd instar larvae devour young leaves or scratch the mesophyll of old leaves. Final instar larva pupates on the underside of leaf (Suppl. material
This species is endemic in Taiwan.
Based on morphology of male genitalia (Fig.
The characters of the male genitalia (Fig.
The Dodona butterfly in Taiwan should be regarded as an endemic species, Dodona formosana, distinct from D. eugenes. The present work and a previous study (
We would like to thank following colleagues for providing specimens and assisting field work: Mr. H.-l. Deng, and A.-m. Li (Chongqing Museum of Natural History, Chongqing); N.-Y. Tsai, J.-H. Lin, L.-H. Wang, J.-Y. Liang, Y.-M. Hsu, K.-W. Hsiao, L. Huang, C.-J. Chang, C.-C. Lu, C.-R. Chen (
Sampling information
Data type: species data
Explanation note: Sample list and sampling information of this study.
Primer information
Data type: molecular data
Explanation note: Primers used in this study.
Phylogenetic trees
Data type: molecular data
Explanation note: Topologies inferred by BI and ML methods based on COI and COII. S3a: BI phylogeny; S3b: ML phylogeny.
Haplotype information
Data type: occurence
Explanation note: Variable sites of 15 Dodona haplotypes among 14 sampled localities in Taiwan.
Morphological genitalia
Data type: species data
Explanation note: Additional male and female genitalia of Dodona formosana. S5a: A Dn032 B Dn039 C Dn064 D Dn067 E Dn082 F Dn026 G Dn059 H Dn083 I Dn090; S5b: Dn055.
Images
Data type: species data
Explanation note: Larval stages of Dodona formosana. A eggs B first instar C second instar D third instar E fourth instar F pupa.